Hydrogenese gene clusters
Hydrogenese gene clusters
High-activity hydrogenase is necessary for our system. To achieve efficient enzymatic activities, we codon-optimized and constructed the whole hydrogenase gene clusters (from Clostridium Acetobutylicum) by leveraging the multi-expression Acembl System. Please refer to Hydrogenase Session for more details.
In this section,we start with a single device of one gene from hydrogenase(From
BBa_K2132004;
BBa_K2132005;
BBa_K2132006;
BBa_K2132007;
BBa_K2132008). A single device including following feagment
- Different resistance gene for better selection.
- Same RBS site to recruit ribosome with equal chance .
- Same T7/Lac promoter to help make the moderate expression level of hydrogenase gene clusters.
- Same cre-loxp recombination site to utilizes Cre recombinase to integrate four basic device into a compositive one.
- BR322 origin in acceptor vs R6k gamma origin in donors to ensure the selection of compositive device and achieve a stable inheritance
Figure 11 Integration of four basic plasmid backbones into one.
We basically relied on the Acembl system for hydrogenases gene cluster construction and finished the cloning of single device with sequencing confirmation.
(Click to see the detail sequenced information:
HydA-SpyCatcher,
HydA-SpyTag,
HydE,
HydF,
HydG)
In using the system, however, we can either fuse 4 single plasmids with one step of Cre recombination or do it step by step, integrating each plasmid one at a time. In order to gain higher success rate, we choose the second way.
We fused pACE-Histag-TEV-HydA-Spytag/pACE-Histag-TEV-HydA-Spycatcher with pDK-HydF together as the first step. To test if we successfully fused the two, we use single restricted endonuclease digestion of XhoI. The restriction gives two bands on a 1% TAE Gel, in accordance with the band predicted by SnapGene®.
Figure 12A Fusion of plasmid 1 and plasmid 4.
Single restricted-endonuclease digestion of Xhol in pACE-Histag-TEV-HydA-Spytag x pDK-HydF gives two bands. The left pic refers to expected results based on SnapGene® software prediction, with two bands at 5427bp and 2146bp, respectively. The right figure refers to the experimental results, which is in good agreement with the software prediction.
Figure 12B Fusion of plasmid 2 and plasmid 4.
Single restricted-endonuclease digestion of Xhol in pACE-Histag-TEV-HydA-Spycatcher x pDK-HydF gives two bands. The left pic refers to expected results based on SnapGene® software prediction, with two bands at 5427bp and 2455bp, respectively. The 2455bp is larger than 2146bp due to the larger SpyCatcher. The right figure refers to the experimental results, which is in good agreement with the software prediction.
Figure 12A/B shows that plasmid1/2 and 4 are successfully fused.
Second step:Fusion of plasmid in step one and plasmid 3.
We test through the selection of LB solid plate with three resistance, Ampicillin, Chloramphenicol, and kanamycin. Then we use single restricted endonuclease digestion of XhoI. There should be two kinds of ways in fusing. Comparing our electrophoresis band with the prediction by SnapGene®, we confirmed the kind we obtained.
Figure 12C Fusion of the plasmid in step one(12A) and plasmid 3.
After the fusion of the plasmid in step one and plasmid 3, there will be one more enzyme restriction site of XhoI. Single restricted-endonuclease digestion of Xhol in pACE-Histag-TEV-HydA-SpyTag x pDK-HydF x pDC-HydE gives two bands. The left pic refers to expected results based on SnapGene® software prediction, with three bands at 5427bp, 2897bp and 2249bp, respectively. The right figure refers to the experimental results, which is in good agreement with the software prediction.
Figure 12D Fusion of the plasmid in step one(12B) and plasmid 3.
After the fusion of the plasmid in step one and plasmid 3, there will be one more enzyme restriction site of XhoI. Single restricted-endonuclease digestion of Xhol in pACE-Histag-TEV-HydA-SpyCatcher x pDK-HydF x pDC-HydE gives two bands. The left pic refers to expected results based on SnapGene® software prediction, with three bands at 5427bp, 2897bp and 2558bp, respectively. The right figure refers to the experimental results, which is in good agreement with the software prediction.
Figure 1C/D shows that plasmids obtained in step 1 and plasmid 3 are successfully fused.
Final step:Fusion of plasmid in step 2 and 5.
This fusion was conferred many possibilities due to the multiple loxP sites that are potentially recognized by Cre, and the fact that some fused loxP sites are reversely separated. However, since the plasmid in step 2 and plasmid 5 are put into the reaction in equal molar, the fully fused plasmid has a better chance. In parallel, we mixed four (plasmid 1/2, 3, 4, 5) plasmids together. After characterization by endonuclease restriction, we obtained the final plasmid. In addition, we find that the mixing of four in one reaction is not efficient.
Figure 12E Fusion of the plasmid in step (12C) and plasmid 3.
For a whole fused plasmid, It becomes hard to analyze it with just Xho I single enzyme. The bar at 3k actually accounts for two bars, with a separation of 20bp. In the picture, although the four bands predicted by SnapGene® can be found on our real gel, it is less clear.
Given the inconvenience with testing by restriction, we turned to resistance screening. The result is that it is resistant to four antibodies (Ampicillin, Chloramphenicol, kanamycin and Spectinomycin).
Expression of the hydrogenase.
As we had successfully get the device,the next step is to induce the expression of the hydrogenase.
To see, we use the antibody of Histag to show the specific of HydA-spycatcher and HydA-spytag and got the result. While we can not avoid the other protein with a similar affinity.
